Investigating mitochondrial dysfunction in neurodegeneration using A Nanoparticle-based Synthetic Mitochondrial DNA (mtDNA) Transcription Regulator

使用基于纳米颗粒的合成线粒体 DNA (mtDNA) 转录调节器研究神经退行性变中的线粒体功能障碍

• 批准号: 10679826
• 负责人: Kibum Lee
• 金额: $ 19.49万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679826
• 关键词: Address; Aftercare; Binding; Blood; Cells; Circulation; Clinic; Cytochrome c Reductase; DNA; DNA Binding; DNA Sequence; DNA delivery; Disease; Electron Transport; Face; Functional disorder; Gene Activation; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Goals; Health; Human; Impairment; Induced pluripotent stem cell derived neurons; Knowledge; Ligands; Light; Link; Mediating; Methods; Mission; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Proteins; Muscle; Mutation; NADH dehydrogenase (ubiquinone); Natural regeneration; Nerve Degeneration; Nerve Regeneration; Neurons; Nuclear; Parkinson Disease; Pathology; Patients; Penetration; Peptides; Performance; Production; Proteins; Public Health; Quality of life; Radiation Protection; Reactive Oxygen Species; Research; Site; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Transcriptional Regulation; Translating; Treatment Efficacy; United States National Institutes of Health; Viral; Viral Vector; base editing; biomaterial compatibility; cytotoxic; disability; disease-causing mutation; factor A; genetic manipulation; genome editing; human disease; immunogenic; improved; injury and repair; innovation; innovative technologies; mitochondrial dysfunction; mitochondrial genome; mtTF1 transcription factor; nanoGold; nanobiomaterial; nanocluster; nanoparticle; nervous system disorder; neuron loss; novel; nuclease; overexpression; prevent; scaffold; small molecule; targeted delivery; technology platform; therapeutic target; tool; transcription factor; uptake

PROJECT SUMMARY Mitochondrial diseases are caused by mutations in genes that encode structural mitochondrial proteins or proteins involved in mitochondrial function. Mitochondrial function abnormalities are among the most common genetic neurological disorders, making them an ideal therapeutic target. The growing knowledge of links between aberrant mitochondrial gene transcription and human diseases critically necessitates an effective approach to controlling mitochondrial DNA (mtDNA) transcription. To this end, developing a method to modulate mtDNA transcription sites specifically is vital for understanding and treating mitochondria-related diseases. However, the Inefficient delivery of DNA binding motifs into mitochondria and difficulty activating mitochondria genes with current technologies limit mitochondrial genome editing/gene manipulation. Advanced techniques for regulating mtDNA transcription have relied on the delivery of exogenous transcription factors, such as mitochondrial transcription factor A (TFAM), DNA oligomers, or DNA-base editing nucleases. Furthermore, translating these advanced tools into therapeutics would require substantial advances in their targeted delivery into mitochondria, as most mitochondrial transcription factors (TFs) and base editing tools face significant hurdles during circulation in blood or other biofluids. Therefore, there is an urgent need to develop novel methods to achieve selective and efficient gene activation in the mitochondria. Addressing the above challenges, the main goal of this proposal is to develop a nanoparticle-based synthetic mitochondrial DNA (mtDNA) transcription regulator to investigate mitochondrial dysfunction in neurodegeneration. The modular MitoScript platform will be assembled from: i) ultra-small fluorescent gold nanoclusters (NC) as scaffolds for assembly of biomolecular ligands; ii) synthetic PIP oligomers as mtDNA binding domains (DBDs) for site-specific mitochondrial transcription regulation; iii) mitochondria-penetrating peptides (MPPs) as mitochondrial localization domains; and iv) a mitochondrial transcription factor motif derived from TFAM as an activation domain (AD). By doing so, we aim to construct an artificial/synthetic mitochondrial TF that can: i) efficiently target mitochondria genes with no cytotoxic effects or immunogenic (e.g., viral vectors) carriers; ii) selectively bind to any target DNA sequences in the mitochondria genome, and iii) controllably downregulate and upregulate mitochondria genes that can eventually alter neural cell fates. We propose to objectively test our central hypothesis and achieve our objectives by addressing the following specific aims: AIM #1 − Construct ND6-targeting MitoScript (ND6-MitoScript) to regulate ND6 genes in mitochondria efficiently; AIM #2 − Validate ND6-MitoScript for ND6 gene overexpression and improved survival in PD patient iPSC- derived neurons; Collectively, we anticipate that our proposed studies will provide an innovative, highly effective, and selective method for developing therapeutic interventions for mtDNA-mediated neurological disorders.

项目摘要 线粒体疾病是由编码结构线粒体蛋白或 参与线粒体功能的蛋白质。线粒体功能异常是最常见的 遗传神经系统疾病，使其成为理想的治疗靶点。越来越了解联系之间的联系 异常的线粒体基因转录和人类疾病至关重要 控制线粒体DNA（mtDNA）转录。为此，开发一种调节mtDNA的方法 转录位点特别对于理解和治疗与线粒体相关的疾病至关重要。但是， 将DNA结合基序递送到线粒体中，并难以激活线粒体基因 当前技术限制了线粒体基因组编辑/基因操纵。确定的高级技术 mtDNA转录在递送外源转录因子（例如线粒体）方面放松 转录因子A（TFAM），DNA低聚物或DNA碱编辑核。此外，翻译这些 治疗的先进工具将需要将其目标交付到线粒体方面取得重大进展， 由于大多数线粒体转录因子（TFS）和基础编辑工具在循环过程中面临重大障碍 在血液或其他生物流体中。因此，迫切需要开发新颖的方法来实现选择性和 线粒体中有效的基因激活。 解决上述挑战，该提案的主要目标是开发基于纳米颗粒的挑战 合成线粒体DNA（mtDNA）转录调节剂，研究线粒体功能障碍 神经变性。模块化的mitoscript平台将从以下方式组装：i）超小荧光金 纳米簇（NC）作为用于生物分子配体组装的支架； ii）合成PIP寡聚物作为mtDNA 位点特异性线粒体转录调控的结合域（DBD）； iii）线粒体渗透 肽（MPP）作为线粒体定位域； iv）线粒体转录因子基序得出 从TFAM作为激活域（AD）。通过这样做，我们旨在构建人工/合成线粒体 可以：i）有效靶向没有细胞毒性作用或免疫原性的线粒体基因（例如，病毒载体） 载体； ii）选择性地结合线粒体基因组中的任何靶DNA序列，并受控 下调和上调线粒体基因，有时会改变神经细胞的命运。 我们建议客观地测试我们的中心假设，并通过解决以下内容来实现我们的目标 具体目的：目标＃1-构建ND6靶向mitoscript（ND6-Mitoscript）以调节线粒体中的ND6基因 有效AIM＃2-验证ND6基因过表达的ND6-Mitoscript和PD患者IPSC-的生存率提高 衍生的神经元；总体而言，我们预计我们的拟议研究将提供创新，高效， 和用于开发MTDNA介导的神经系统疾病的治疗干预措施的选择性方法。